The present invention relates to an apparatus suitable for column or slab gel electrophoresis and destaining. More particularly, it relates to a gel electrophoresis cell unit wherein the temperature within a gel medium can be controlled to prevent overheating. This is particularly important for the electrophoresis of biological samples wherein overheating can cause loss in viability.
Gel electrophoresis was explored in the early sixties, by L. Orstein and B. J. Davis using polyacrylamide gels. Since then, the method developed by Orstein and Davis has been applied extensively in the separation of chemical mixtures and identification of their components, particularly for the separation and identification of proteins and nucleic acids in biological fluids.
Vertical gel electrophoresis, one form of gel electrophoresis, is a process for the separation of components in a sample mixture by the application of an electric potential over the length of a gel medium held vertically in a glass tube or channel to cause various components in a sample mixture deposited at one end of a gel medium to differentially migrate over the length of the gel medium. A vertical gel electrophoresis apparatus has mainly two parts: a power supply and an electrophoresis cell unit. The electrophoresis cell unit comprises: a first or upper buffer chamber, a second or lower buffer chamber, an upper electrode, a lower electrode and a plurality of vertically disposed gel columns or slabs having opposite ends opening into the upper and the lower buffer chambers. The upper ends of the gel columns or slabs are in contact with a first or upper buffer solution in the first or upper buffer chamber, and the lower ends of the gel columns or slabs are in contact with a second or lower buffer solution in the second or lower buffer chamber. An electric potential is applied across the ends of the gel columns or slabs by the use of electrodes connected to the power supply and placed separately in the upper and lower buffer solutions.
Until recently, conventional electrophoresis cell units are basically of the same design, employing a holder for gel tubes which also serves as the bottom of the upper buffer chamber. Sealing gaskets on the gel tube holder further serves to separate the buffer solutions in the upper and lower buffer chambers, so that there is no conductance of electricity between the upper and lower buffer solutions except through the gel tubes.
Because of this design, there are several problems. Firstly, the rubber gaskets are inserted into holes on the holder which serves also as the bottom of the upper buffer chamber. This means that the upper buffer chamber must be emptied and inverted for the insertion or extraction of gel tubes. Secondly, the lower ends of all gel tubes held on the holder are immersed in the lower buffer solution simultaneously. This leads to formation of air bubbles at the lower end of the gel tubes. The air bubbles obstruct the flow of electric current through the gel columns, and must be eliminated. However, elimination of all of the air bubbles is troublesome. Thirdly, rubber gaskets, used to hold the gel tubes and to act as seals separating the upper and lower buffer solutions, surround and contact a portion of the gel tubes where the sample gel is usually loaded onto the column. In this portion of the gel tube, the concentration of the sample is relatively high, and is, therefore, more sensitive to heat. The rubber gaskets, being good heat insulators, do not allow a sample in that portion of the tube surrounded by the rubber gasket to be cooled sufficiently. This causes the samples to overheat with concomitant loss of viability, especially in biological samples. Fourthly, the two buffer solutions are usually at different levels which leads to a difference in hydrostatic pressure between upper and lower ends of the gel tubes and also induces an underflow in the gel medium. This disturbs the electrophorectic behavior of the samples, sometimes even causing a low concentration gel to slide out of its supporting glass tube.
In Hoefer, U.S. Pat. No. 3,867,271, an attempt was made to modify the conventional construction of tube gel electrophoresis cell units. The main emphasis was on cooling the gel tubes by inclusion of a central cooling chamber. However both end portions of each gel tube were supported by rubber gaskets. This resulted in less cooling of the gel column than expected. Further, the Hoefer design with its central cooling chamber and provisions for circulating water therein, is complicated, making it hard to manufacture and inconvenient to operate. Moreover, the Hoefer design did not totally overcome the problems in conventional electrophoresis cells units such as: overheating, the formation of air bubbles, or hydrostatic inequilibrium. In fact, two years later, Hoefer, in U.S. Pat. No. 4,048,049, reverted to a more conventional design. The central cooling chamber of his prior design was replaced with a cooling core; the tubes were held in the middle with rubber gaskets. These modifications still did not overcome the problems of bubble formation or hydrostatic inequilibrium and did not completely eliminate the problems of overheating over the entire length of the gel tube. Recently issued U.S. Pat. No. 4,284,491 to Vesterberg, discloses an electrophesis cell unit which is basically the same as Hoefer U.S. Pat. No. 3,867,271 except that the unit has a rectangular rather than a round configuration. Therefore, the various problems discussed above also apply to the Vesterberg design.
Vertical slab gel electrophoresis cells have also followed the conventional design of tube gel electrophoresis cells. As a result, all of the defects described above also applies to vertical slab gel electrophoresis cells. See, for example, U.S. Pat. Nos. 3,932,265; 3,719,580; 4,142,960; 4,290,871; 4,292,161; and 4,224,134.
It is, therefore, an object of the present invention to provide a novel tube gel electrophoresis cell which is simple to manufacture and convenient to operate.
Another object of the invention is to provide an apparatus for vertical gel electrophoresis wherein the gel column will be cooled adequately throughout the migration of the sample on the column to prevent denaturation or loss of viability due to overheating.
A further object of the invention is to provide an apparatus for tube gel electophoresis which is easily convertible to an electrophoresis or diffusion destainer.